Detection of isoniazid resistent strains of M. tuberculosis

ABSTRACT

A method for determining the susceptibility of a strain of M. tuberculosis to isoniazid is provided comprising employing the techniques of restriction length polymorphism analysis to determine whether or not the DNA of said strain has an NciI-MspI restriction site at the codon corresponding to codon 463 of an M. tuberculosis katG gene consensus sequence.

BACKGROUND OF THE INVENTION

Despite more than a century of research since the discovery of Mycobacterium tuberculosis, the aetiological agent of tuberculosis, this disease remains one of the major causes of human morbidity and mortality. There are an estimated 3 million deaths annually attributable to tuberculosis (see, D. Snider, Rev. Inf. Dis., S335 (1989)), and although the majority of these are in developing countries, the disease is assuming renewed importance in the West due to the increasing number of homeless people and the impact the AIDS epidemic (see, R. E. Chaisson et al., Am. Res. Resp. Dis., 23, 56 (1987); D. E. Snider, Jr. et al., New Engl. J. Med., 326, 703 (1992); M. A. Fischl et al., Ann. Int. Med., 117, 177 (1992) and ibid. at 184.

Isonicotinic acid hydrazide or isoniazid (INH) has been used in the treatment of tuberculosis for the last forty years due to its exquisite potency against the members of the "tuberculosis" groups--Mycobacterium tuberculosis, M. bovis and M. africanum (G. Middlebrook, Am. Rev. Tuberc., 69, 471(1952) and J. Youatt, Am. Rev. Resp. Dis., 99, 729 (1969)). Neither the precise target of the drug, nor its made of action are known, but INH treatment results in the perturbation of several metabolic pathways of the bacterium. However, shortly after its introduction, INH-resistant isolates of Mycobacterium tuberculosis emerged. See M. L. Pearson et al., Ann. Int. Med, 117, 191 (1992) and S. W. Dooley et al., Ann Int. Meal, 117, 257 (1992).

Several investigators have associated the toxicity of INH for mycobacteri with endogenous catalase activity. See, for example, "Isonicotinic acid hydrazide," in F. E. Hahn, Mechanism of Action of Antibacterial Agents, Springer-Verlag (1979) at pages 98-119. This relationship was strengthened by a recent report by Ying Zhang and colleagues in Nature, 358, 591 (1992) which described the restoration of INH susceptibility in an INH resistant Mycobacterium smegmatis strain after transformation using the catalase-peroxide (katG) gene from an INH sensitive M. tuberculosis strain. In a follow-up study, Zhang and colleagues in Molec. Microbial., 8, 521 (1993) demonstrated the restoration of INH susceptibility in INH resistant M. tuberculosis strains after transformation by the functional katG gene. As reported by B. Heym et al., J. Bacteriol., 175, 4255 (1993), the katG gene encodes for a 80,000-dalton protein.

A significant problem in control of multiple drug resistant tuberculosis (MDR-TB) epidemics has been the delay in the detection of drug resistance. Currently laboratory methods used to identify M. tuberculosis drug resistance require weeks to months for results. The rapid detection of M. tuberculosis directly from clinical samples has been possible recently by virtue of the availability of polymerase chain reaction (PCR) and the recognition of diagnostic sequences amplified by the appropriate primers. The ability to conduct PCR analyses depends on having a high enough gene or gene product concentration so that the molecular tools work efficiently even when the organism numbers are low. Thus, the most efficient molecular assays used to detect M. tuberculosis depend on the IS6110 insertion sequence (about 10 copies) or the 16S ribosomal RNA (thousands of copies). See, respectively, K. D. Eisenach et al., J. Infect. Dis., 61, 997 (1990) and N. Miller et al., Abstracts ASM, Atlanta, Ga. (1993) at page 177. Recently, B. Heym et al. (PCT WO 93/22454) disclose the use of polymerase chain reaction to amplify portions of the katG gene of putative resistant strains. The PCR products were evaluated by single-strand conformation polymorphism (SSCP) analysis, wherein abnormal strand motility on a gel is associated with mutational events in the gene. For example, in five strains, a single base difference was found in a 200 bp sequence, a G to T transversion at position 3360.This difference would result in the substitution of Arg-461 by Leu. However, carrying out SSCP on a given clinical sample can be a laborious procedure that requires sequencing to conform whether mutatiom or deletions predictive of drug resistance are in fact present in the target gene.

There is a continuing need in the art to develop a simple test permitting the rapid identification of INH-resistant strains of M. tuberculosis.

SUMMARY OF THE INVENTION

The present invention provides a method to rapidly identify strains of M. tuberculosis which are resistant to isoniazid (INH). The method is based on the identification of an NciI-MspI restriction site in codon 463 of a consensus sequence determined for the katG gene of M. tuberculosis which is absent in the corresponding codon in a number of INH resistant strains. The determination is preferably made by employing the techniques of restriction length polymorphism analysis. Therefore, in one embodiment, the present assay comprises the steps of:

(a) amplifying a portion of the katG gene of an M. tuberculosis isolate to yield a detectable amount of DNA comprising a plurality of NciI-MspI restriction sites;

(b) cleaving the DNA with a restriction endonuclease at said sites to yield DNA fragments; and

(c) employing the techniques of gel electrophoresis to determine whether the number and location of the fragments is indicative of the absence of an NciI-MspI restriction site in codon 463 of said katG gene, wherein said absence is indicative of an INH-

resistant swain of M. tuberculosis.

More specifically, gel electrophoresis is employed to compare the number and location of the DNA fragments to the number and location of DNA fragments derived from cleavage of DNA derived from an equivalent portion of the katG gene wherein the NciI-MspI restriction site at codon 463 is present, wherein a determination of the absence of the restriction site at codon 463 in the katG gene is indicative of an INH resistant swain of M. tuberculosis.

Preferably, the control DNA sequence of the portion of the katG gene wherein the codon 463 restriction site is present corresponds to a portion of SEQ ID NO:1 (FIG. 1, upper sequence). As discussed below, such a portion of DNA can be derived from strain H37Rv MC. The assay of step (c) also preferably includes negative control DNA fragments derived from an INH resistant strain which does not include the codon 463 NciI-MspI restriction site in the katG gene.

The term "equivalent" is defined herein to mean that any two portions of the katG gene would comprise the same number of NciI-MspI sites if the portions both were selected from a portion of the DNA of SEQ ID NO:1, and that the portions do not differ in size before cleavage to the extent that the number of fragments obtained cannot be compared following side-by-side gel electrophoresis and visualization of the resultant fragments, as described hereinbelow. Preferably, the control DNA for step (c) comprises five NciI-MspI restriction sites in each DNA molecule prior to cleavage, and the DNA of step (a) comprises four NciI-MspI restriction sites in each DNA molecule, prior to cleavage. Preferably, the portion of the katG locus which is amplified is a minor portion of the entire katG gene, i.e., about 40-70%, and is isolated and amplified by polymerase chain reaction, as described hereinbelow. The term "location" refers to the Rf of a given fragment on the gel.

The present invention also provides oligonucleotides useful in pairs as primers to initiate the polymerase chain reaction (PCR). PCR is useful both to amplify katG DNA so as to prepare both the target DNA of step (a) of the present process, as well as the DNA which is used to prepare the control digest of step (c).

The present invention also provides isolated, purified DNA corresponding to the consensus sequence derived for M. tuberculosis katG gene. This DNA was found to occur in nature as the katG gene of M. tuberculosis strain H37Rv MC, as maintained at the Mayo Clinic. The present invention also includes isolated, purified DNA encoding the consensus amino acid sequence encoded by the consensus katG DNA, as well as equivalent DNA sequences which also encode this amino acid sequence (a consensus catalase peroxidase polypeptide) and will provide the isolated, purified polypeptide corresponding to the consensus amino acid sequence.

This polypeptide can be prepared by expression by bacteria, yeast or insect cells transformed with the DNA sequences of the present invention, operatively linked to regulatory regions functional in the transformed host cells. The polypeptide can be used as a standard M. tuberculosis catalase peroxidase, to correlate enzymatic activity (relative level, loss and restoration), with INH modification and degradation and drug resistance in M. tuberculosis.

The present invention also provides a kit comprising, separately packaged in association:

(a) a pair of oligonucleotide primers selected so as to amplify a portion of the DNA of the M. tuberculosis katG gene comprising a plurality of NciI-MspI restriction sites, i.e. 2-5 restriction sites;

(b) an amount of a restriction endonuclease such as NciI, MspI or a mixture thereof, effective to cleave the amplified portion of said DNA at the NciI-MspI restriction sites.

The present kits will also preferably comprise instruction means for carrying out the present assay, i.e., a printed package insert, tag or label, or an audio or video tape. The present kits will also preferably comprise a control DNA digest prepared by amplifying a portion of the consensus DNA of SEQ ID NO:1 (FIG. 1), that is essentially identical to the portion defined and amplified by the pair of primers, followed by digestion of the DNA with a suitable restriction endonuclease such as NciI, MspI or a mixture thereof.

Although the present invention is exemplified by the use of NciI digestion, the restriction endonuclease depicted on Table 1 can also be employed.

                  TABLE 1                                                          ______________________________________                                         Specificity       RE                                                           ______________________________________                                         Cuts 463-R (sensitive):                                                                          CCSGG.sup.a CCGG                                                               NciI        MspI                                                               AhaI        HapII                                                                          HpaII                                            Cuts 463-L (resistant):                                                                          CCWGG.sup.a                                                                    ApyI                                                                           BstNI                                                                          EcoRII                                                                         MvaI                                                         ______________________________________                                          .sup.a S = C or G;                                                             W = A or T                                                               

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, panels A-D, depicts the consensus DNA sequence of the katG gene as the upper of the pair of sequences (61-2295) (SEQ ID NO:1). This DNA sequence data has been submitted to Gen Bank and has been assigned accession number UO6262. The lower of the pair of sequences depicts nucleotide sequence 1970-4190 of the KpnI fragment bearing the katG gene as depicted in FIG. 6 of Institut Pasteur et al. (published PCT application WO 93/22454). This sequence (SEQ ID NO:2) has been deposited in the EMBL data library under accession number X68081 (Gen Bank X68081. gb₋₋ ba). Dots (.) above the sequence mark every tenth base. The upper sequence is in lower case in areas where variation in the sequence among isolates described hereinbelow and the consensus sequence was found. The arrow before position 70 and after position 2291 of the upper sequence indicate the sequence of the katG gene.

FIG. 2 depicts the katG amino acid consensus sequence derived from 15 strains of M. tuberculosis (SEQ ID NO:7).

FIG. 3 schematically depicts the NciI restriction sites for the part B amplicon of katG. The (*) depicts the site of the Arg→Leu mutation which is found in some INH resistant M. tuberculosis strains.

FIG. 4 depicts the results of a gel electrophoresis of the NciI digest of the part B amplicon of 14 strains of M. tuberculosis (1-14).

DETAILED DESCRIPTION OF THE INVENTION

Wild type strains of M. tuberculosis are highly susceptible to INH with minimum inhibitory INH concentration (ICmin) ≦0.02 μg/ml and a susceptible strain is considered to be one with an IC <1.0 μg/ml. At the Mayo Clinic, Rochester, Minn., clinical strains of M. tuberculosis (including MDR-TB strains) were identified which exhibit intermediate to high level resistance to INH (ICmin range 1.0 to >32 μg/ml). Many of these strains, especially those highly resistant to INH (≧1.0 μg/ml), exhibited diminished catalase activity as assessed by a semiquantitative technique. The mean semiquantitative catalase was 16.5 mm for 6/15 strains with INH ICmin <1.0 μg/ml and 13.3 mm for 9/15 strains with ICmin ≧1.0 μg/ml. To develop the present assay, it was first necessary to determine whether some M. tuberculosis strains have decreased INH sensitivity as a result of katG gene mutations. Therefore, the nucleic acid sequences of the katG genes for both INH (ICmin <1.0 μg/ml) and INH resistant (ICmin ≧1.0 μg/ml) M. tuberculosis strains were determined. From the DNA sequencing data generated, a katG consensus sequence was derived, and katG sequences from all 15 M. tuberculosis strains (INH sensitive and INH resistant) were compared to the consensus sequence to determine katG deviations. These data in turn revealed a common restriction fragment polymorphism (RFLP) in nearly half (44%) of the 43 INH resistant strains examined.

Five of nine INH resistant strains (INH ICmin ≧1.0 μg/ml) had one or more missense mutations; one had a nonsense mutation; one had an 8 base pair deletion and one had no mutations in the coding sequences. All of the five strains with missense mutations had a common G to T transversion in the 463 codon causing replacement of arginine with leucine and loss of an NciI-MspI restriction site. Six INH sensitive strains (INH ICmin <1.0 μg/ml) were also sequenced and found to have from none to 11 amino acid differences with the consensus sequence of all 15 strains, but none of the mutations affected codon 463 or its overlapping restriction site. Restriction analysis of a total of 32 sensitive and 43 resistant strains, showed that 19 of 43 (44%) of all INH resistant strains had lost the NciI-MspI restriction site at the locus of codon 463 while only 1 of 32 sensitive strains had this restriction polymorphism.

These results indicate that the mutation, arginine-→leucine, in the codon 463 of the M. tuberculosis catalase-peroxidase (katG) gene occurs in a significant fraction (44%) of INH resistant M. tuberculosis strains (INH ICmin ≧1.0 μg/ml). Furthermore, this mutation can be determined using a rapid relatively simple method, i.e., PCR amplification, digestion and monitoring for a loss of an NciI-MspI restriction site by RFLP, as described in detail hereinbelow. Although in a preferred embodiment of the invention, the number and location of the fragments is determined by gel electrophoresis, the presence or absence in the digest of a fragment comprising the NciI-MspI restriction can be determined by other methods known to the art, including immunoassays (dot blots and reverse dot blots), DNA probes, microtiter well capture and the like.

The present invention will be further described by reference to the following detailed examples, wherein 58 clinical strains of Mycobacterium tuberculosis were obtained from the Mycobacteriology Laboratory at the Mayo Clinic, Rochester, Minn., and 17 M. tuberculosis DNA preparations were obtained from the GWL Hansen's Disease Center, Louisiana State University, Baton Rouge, La. The strain designated H37Rv MC has been maintained at the Mayo Clinic for over 50 years, and therefore was isolated before INH became available as a treatment modality for tuberculosis (circa 1952). H37Rv was deposited in the American Type Tissue Collection, Rockville, Md. in 1937 by A. Karlson of the Mayo Clinic under the accession number ATCC 25618, and has been freely available to the scientific community since. An apparent variant of this strain is disclosed in PCT WO 93/22454 (SEQ ID NO:2, herein). The ATCC strains 27294 and 25618 were recovered from the same patient in 1905 and 1934, respectively. All clinical M. tuberculosis strains were confirmed as M. tuberculosis using routine identification techniques described by J. A. Washington, "Mycobacteria and Norcardia," in: Laboratory Procedures in Clinical Microbiology, 2d ed., Springer-Verlag, NY (1985) at pages 379-417.

For the 15 M. tuberculosis strains for which complete katG DNA sequencing was performed, susceptibility testing was done at the Mayo Clinic using Middlebrook 7H11 agar (DiMed, Inc., St. Paul, Minn. 55113) and the 1% proportion method described in Manual of Clinical Microbiology, 5th ed., A. Balows et al., eds., Amer. Soc. Microbial. (1991) at pages 1138-52. The same method was used at the Mayo Clinic to determine susceptibility for an additional 43 M. tuberculosis strains for which restriction fragment length polymorphisms (RFLP) were determined. Isoniazid concentrations tested using this method included: 0.12, 0.25, 1.0, 2, 4, 8, 16, 32 μg/ml for the 15 strains sequenced and 1.0 and 4.0 μg/ml for th eremaining 45 strains. Isoniazid resistance was defined as a maximum inhibitory concentration (ICmin) ≧1.0 μg/ml. Susceptibility testing was performed elsewhere for an additional 17 M. tuberculosis strains for which DNA lysates were provided by Diana L. Williams, Baton Route, La. These strains were of diverse geographical origin. 10 of these 17 strains, originated from Japan. The remaining 7 M. tuberculosis INH resistant strains included multiple drug resistant strains from recent MDR-TB nosocomial epidemics in New York, N.Y. and Newark, N.J. All were INH resistant (ICmin ≧1.0 μg/ml), and had resistance to at least one other drug. For all strains provided by Williams, the 1% direct proportion method was used, but the concentration of INH tested, and the media used varied as to site.

To conduct a semiquantitative test of catalase activity, M. tuberculosis strains were propagated on Lowenstein-Jensen media deeps contained in 20×150 mm screw-capped tubes. One ml of a 30% hydrogen peroxide (EM, Science, Gibbstown, N.Y. 08027) and 10% Tween 80 (Aldrich Chemical Co., Milwaukee, Wis. 53233) solution mixed in a 1:1 ratio was applied to the surface of growth. After 5 minutes, the highest (mm) of the column of bubbles (O₂) generated was recorded.

EXAMPLE 1 DNA Isolation and Polymerase Chain Reaction

For M. tuberculosis strains obtained from Mayo Clinic samples, DNA was extracted from cells using phenol (Boehringer Mannhaim, Indianapolis, Ind. 46250-0414) and TE (1.0M Tris HCl pH 8.0, 0.1M EDTA, Sigma, St. Louis, Mo. 63778) in a ratio of 600μl:400 μl and 0.1 mm zirconium beads (Biospec Products, Bartlesville, Okla. 74005). The mixture was processed in a mini-bead beater for 30 seconds and allowed to stand for an additional 15 minutes. Following a brief centrifugation to sediment the zirconium beads, DNA in the supernatant was extracted using the IsoQuick kit (MicroProbe Corp., Garden Grove, Calif. 92641).

The DNA sequence for katG (EMBL no. X6808124) employed to design primers is depicted in FIG. 1 (A-D), lower strand. The PCR method of R. K. Saiki et al., Science, 239, 487 (1988) was used to amplify the katG gene (ca. 2220 base pairs) in two segments which were designated A and B. Genomic DNA preparations (2 μl) were used with primers A1 (5' ##STR1##

The PCR mixture (50 μl) contained 10 mM TRIS, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM each of dATP, dTIP, dGTP, dCTP, 1 μM of each primer pair, 10% glycerol, 1.25 units/50 μl AmpliTaq DNA polymerase (Perkin Elmer Cetus). The mixture was overlaid with mineral oil and subjected to 4 min at 95° C. followed by 50 cycles of 1 min at 94° C. and 2 min at 74° C. A 1495 base pair product from the first half of katG was generated from the A1-A4 primers and 1435 base pair product was generated with the B1-B2 primer pair.

EXAMPLE 2 DNA Sequencing and Homology Analysis

The polymerase chain reaction (PCR) products were prepared for sequencing using the Magic™ PCR Preps DNA Purification System (Promega Corp., Madison, Wis. 53711). The DNA sequences were determined in both directions using the Taq dye-deoxy terminator cycle sequencing kit and 373A DNA sequencer (Applied Biosystems, Foster City, Calif. 94404) using a series of internal sequencing primers which provided appropriate coverage of katG.

The sequence data were analyzed using version 7 of the Genetics Computer Group sequence analysis software, as disclosed by J. Devereux et al., Nucl. Acids Res., 12., 387 (1984). From the 15 M. tuberculosis DNA sequences, a consensus sequence was derived to which all M. tuberculosis strains were compared. This consensus sequence is depiceed in FIG. 1 (A-D) as the upper strand, and is compared to the sequence for katG (EMBL no. X6808124), depicted as the lower strand. The two sequences have 98.6% identity, as determined by the GCG program BESTFIT. The DNA sequence data has been submitted to Gen Bank and can be referenced by the accession numbers UO6262 (H37Rv MC), UO6258 (ATCC 25618), UO6259 (ATCC 27294), UO6260 (G6108), UO6261 (H35827), UO6270 (L6627-92), UO6271 (L68372), UO6264 (L11150), UO6268 (L24204), UO6269 (L33308), UO6265 (L16980), UO6266 (L1781), UO6272 (TMC306), UO6263 (L10373), and UO6267 (L23261).

The DNA data was then translated, aligned for comparison and a consensus amino acid sequence was generated (FIG. 2) (SEQ ID NO:7).

In general, the overall sequence agreement between INH sensitive and resistant strains was very high; the only deviation are those shown in Table 2.

                                      TABLE 2                                      __________________________________________________________________________     Analysis of Catalase-Peroxidase (katG) Gene in M. tuberculosis                 __________________________________________________________________________     Strains                                                                               INH                                                                            resistance                                                                     (μg/ml) Amino Acid (Codon).sup.b                                     Strain INH   Catalase                                                                            2  10  18 19  53 65 66  90 126 128                                                                               169 224 243                __________________________________________________________________________     H37Rv MC                                                                              <0.12 20                                                                ATCC 25618                                                                            <0.12 12          N--S                                                                              G--D                                                                               A--P                                                                              A--T                                                                              A--P              Q--E                   ATCC 27294                                                                             0.12 28   P--S   N--S                                                                              G--D                        Q--E                                                                               A--S               G6108  <0.12 12                                                                H35827  0.25 14   P--S   N--S                                                                              G--D   A--T                                                                              A--P   M--I           A--S               L6627-92                                                                              0.5   13                                  R--Q                          L68372  1     8   P--S   N--S                M--I   G--A                                                                               Q--E                   L11150  8    28                                                                L24204  8    36                                                                L33308  8    15                                                                L16980 16    15                                                                L1781  32     5                                                                TMC 306                                                                               >32    5                           W*.sup.c                             L10373 >32    5      8 bpd.sup.d                                               L23261 >32    5                                                                             Consensus                                                                           P      N  G   A  A  A   W  M   R  G   Q   A                  __________________________________________________________________________            INH                                                                            resistance                                                                     (μg/ml)                                                                          Cata-                                                                              Amino Acid (Codon).sup.b                                       Strain INH  lase                                                                               258                                                                               264 281 302                                                                               315                                                                               337                                                                               424                                                                               429                                                                               444 463                                                                               505 550 589                                                                               609                __________________________________________________________________________     H37Rv MC                                                                              <0.12                                                                               20                                                                 ATCC 25618                                                                            <0.12                                                                               12                                              M--I               ATCC 27294                                                                             0.12                                                                               28                      A--E                                                                              P--S                                    G6108  <0.12                                                                               12                            A--V       A--D                      H35827  0.25                                                                               14  N--S                                                                              A--T          Y--F                       M--I               L6627-92                                                                              0.5  13                                                                 L68372  1    8     A--V                                                                               A--V      Y--C         R--L                             L11150  8   28             S--R                                                                              S--T                                             L24204  8   36                                R--L                             L33308  8   15                                                                 L16980 16   15                S--T                                             L1781  32    5     A--T                       R--L       P--T                                                                              M--I               TMC 306                                                                               >32   5                                                                 L10373 >32   5                                                                 L23261 >32   5                   Y--F         R--L                                                                              W--R       M--I                           Con-                                                                               N  A   A   S  S  Y  A  P  A   R  W   A   P  M                              sensus                                                             __________________________________________________________________________      *1Cmin denotes maximum inhibitory concentration, INH = isoniazid, RIF          rifampin, ETHAM ethambutol, STR streptomycin, CIP ciptrofloxacin               .sup.b A denotes alanine, C cysteine, D aspartic acid, E glutamic acid, F      phenylalanine, G glycine, I isoleucine, K lysine, L leucine, M methionine      N asparginine, P proline, Q glutamine, R arginine S serine, T threonine,       valine, H tryptophan, Y tryosine, 8 bpd 8 base pair deletion                   .sup.c TGG→TGA (U→stop codon)                                    .sup.d base pair deletion corresponding to wild type coordinates 98-105        creates a new TAG stop codon beginning 11 bp from corrdinate 97.         

The data in Table 2 show that only two strains, H37Rv MC and L3308, are completely homologous to the consensus. They are INH sensitive (INH ICmin <1.0 μg/ml) and INH resistant (ICmin ≧1.0 μg/ml), respectively. All other strains listed in Table 2 had 1 to 11 differences with the consensus and there was no strong correlation between the number of differences and INH sensitivity. In fact, the INH sensitive strains had the most deviations.

In the group of INH resistant strains, the most frequent change observed was the conversion of arginine at codon 463 to leucine. This was detected in five of nine isolates examined. There was not a consistent correlation between the loss of catalase activity and INH resistance since strains L11150 and L24204 had high levels of enzymatic activity, yet were INH resistant. Moreover, several other INH resistant strains showed catalase activity near the mean activity (16.5 mm) of the sensitive strains. Two other isolates had lost the ability to make normal katG gene product due either to an eight bp deletion (L10373, semiquantitative catalase, 3 mm) or a nonsense mutation (TMC 306, semiquantitative catalase 5 mm). It was not possible to determine if, or how, any of the deviations from the consensus reported in Table 2 affect catalase activity or cause INH resistance. However, the change at codon 463 is frequent enough that is indicative of resistance.

The DNA sequence analysis indicated that the codon 463 occurs in the context of an NciI-MspI restriction site (both enzymes recognize the same site). Thus, when in the wild type sequence depicted in FIG. 1 at bases 1455-1458, CCGGG, is changed to CCTGG, it is no longer recognized (or cleaved) by either of these enzymes. The 1435 bp amplicon produced from the half of KatG gene containing codon 463 normally has five NciI-MspI restriction sites whereas the codon altered strains have only four sites, as shown in FIG. 3. The loss of the site in question causes a unique restriction fragment length polymorphism (RFLP), which can be readily adapted to assay for resistant strains, as described in Example 3, below.

EXAMPLE 3 RFLP Analysis

For restriction fragment length polymorphism (RFLP) analysis, a 1435 base pair amplimer (produced using the B1-B2 primers) representing the 3' half of the katG gene was generated using PCR and then digested with NciI or MspI (Sigma Chemical Co., St. Louis, Mo. 63178). The gene fragments were analyzed with agarose gel electrophoresis using 2% Metaphor agarose (FMC BioProducts, Richland, Me. 04811). The gel was stained with ethidium bromide and photographed. The investigator who performed all restriction digests and electrophoresis was blinded as to the INH ICmin results.

The results of this experiment are depicted in FIG. 4, wherein Lane 1 denotes strain H37Rv MC, ICmin=<0.12 μg/ml; (2) L6627-92, 0.5 μg/mL; (3) L68372, 1.0 μg/ml; (4) L16980, 16 μg/mL; (5) L39791, 16 μg/mL; (6) L1781, 32 μg/mL; (7) L9118, 4 μg/mL; (8) L11150, 8 μg/mL; (9) L24204, 8 μg/mL; (10) L68858, <0.12 μg/mL; (11) 1115A <0.12 μg/mL; (12) L23261, >32 μg/mL; (13) 1341, >32 μg/mL; (14) M10838, >32 μ/mL; (15) molecular weight standard: PCR markers (United States Biochemical Corp., Cleveland, Ohio 44122). The digests obtained from resistant strains can be readily visually detected and differentiated from digests from susceptible strains.

Subsequently, a total of 75 M. tuberculosis strains (including the 15 strains sequenced) were analyzed for their loss of the appropriate restriction site. Of these strains, 32 were INH sensitive and 43 were INH resistant. The data showed that 19 (44%) of the 43 resistant strains had lost the expected restriction site in codon 463. One of the 33 (2.9%) sensitive strains had lost this restriction sites as well. None of the six sensitive strains listed in Table 1 lost this site.

All publications, patents and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 7                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2235 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        AGGAATGCTGTGCCCGAGCAACACCCACCCATTACAGAAACCACCACCGGAGCCGCTAGC60                 AACGGCTGTCCCGTCGTGGGTCATATGAAATACCCCGTCGAGGGCGGCGGAAACCAGGAC120                TGGTGGCCCAACCGGCTCAATCTGAAGGTACTGCACCAAAACCCGGCCGTCGCTGACCCG180                ATGGGTGCGGCGTTCGACTATGCCGCGGAGGTCGCGACCATCGACGTTGACGCCCTGACG240                CGGGACATCGAGGAAGTGATGACCACCTCGCAGCCGTGGTGGCCCGCCGACTACGGCCAC300                TACGGGCCGCTGTTTATCCGGATGGCGTGGCACGCTGCCGGCACCTACCGCATCCACGAC360                GGCCGCGGCGGCGCCGGGGGCGGCATGCAGCGGTTCGCGCCGCTTAACAGCTGGCCCGAC420                AACGCCAGCTTGGACAAGGCGCGCCGGCTGCTGTGGCCGGTCAAGAAGAAGTACGGCAAG480                AAGCTCTCATGGGCGGACCTGATTGTTTTCGCCGGCAACTGCGCGCTGGAATCGATGGGC540                TTCAAGACGTTCGGGTTCGGCTTCGGCCGGGTCGACCAGTGGGAGCCCGATGAGGTCTAT600                TGGGGCAAGGAAGCCACCTGGCTCGGCGATGAGCGTTACAGCGGTAAGCGGGATCTGGAG660                AACCCGCTGGCCGCGGTGCAGATGGGGCTGATCTACGTGAACCCGGAGGGGCCGAACGGC720                AACCCGGACCCCATGGCCGCGGCGGTCGACATTCGCGAGACGTTTCGGCGCATGGCCATG780                AACGACGTCGAAACAGCGGCGCTGATCGTCGGCGGTCACACTTTCGGTAAGACCCATGGC840                GCCGGCCCGGCCGATCTGGTCGGCCCCGAACCCGAGGCTGCTCCGCTGGAGCAGATGGGC900                TTGGGCTGGAAGAGCTCGTATGGCACCGGAACCGGTAAGGACGCGATCACCAGCGGCATC960                GAGGTCGTATGGACGAACACCCCGACGAAATGGGACAACAGTTTCCTCGAGATCCTGTAC1020               GGCTACGAGTGGGAGCTGACGAAGAGCCCTGCTGGCGCTTGGCAATACACCGCCAAGGAC1080               GGCGCCGGTGCCGGCACCATCCCGGACCCGTTCGGCGGGCCAGGGCGCTCCCCGACGATG1140               CTGGCCACTGACCTCTCGCTGCGGGTGGATCCGATCTATGAGCGGATCACGCGTCGCTGG1200               CTGGAACACCCCGAGGAATTGGCCGACGAGTTCGCCAAGGCCTGGTACAAGCTGATCCAC1260               CGAGACATGGGTCCCGTTGCGAGATACCTTGGGCCGCTGGTCCCCAAGCAGACCCTGCTG1320               TGGCAGGATCCGGTCCCTGCGGTCAGCCACGACCTCGTCGGCGAAGCCGAGATTGCCAGC1380               CTTAAGAGCCAGATCCGGGCATCGGGATTGACTGTCTCACAGCTAGTTTCGACCGCATGG1440               GCGGCGGCGTCGTCGTTCCGTGGTAGCGACAAGCGCGGCGGCGCCAACGGTGGTCGCATC1500               CGCCTGCAGCCACAAGTCGGGTGGGAGGTCAACGACCCCGACGGGGATCTGCGCAAGGTC1560               ATTCGCACCCTGGAAGAGATCCAGGAGTCATTCAACTCCGCGGCGCCGGGGAACATCAAA1620               GTGTCCTTCGCCGACCTCGTCGTGCTCGGTGGCTGTGCCGCCATAGAGAAAGCAGCAAAG1680               GCGGCTGGCCACAACATCACGGTGCCCTTCACCCCGGGCCGCACGGATGCGTCGCAGGAA1740               CAAACCGACGTGGAATCCTTTGCCGTGCTGGAGCCCAAGGCAGATGGCTTCCGAAACTAC1800               CTCGGAAAGGGCAACCCGTTGCCGGCCGAGTACATGCTGCTCGACAAGGCGAACCTGCTT1860               ACGCTCAGTGCCCCTGAGATGACGGTGCTGGTAGGTGGCCTGCGCGTCCTCGGGCAAACT1920               ACAAGCGCTTACCGCTGGGCGTGTTCACCGAGGCCTCCGAGTCACTGACCAACGACTTCT1980               TCGTGAACCTGCTCGACATGGGTATCACCTGGGAGCCCTCGCCAGCAGATGACGGGACCT2040               ACCAGGGCAAGGATGGCAGTGGCAAGGTGAAGTGGACCGGCAGCCGCGTGGACCTGGTCT2100               TCGGGTCCAACTCGGAGTTGCGGGCGCTTGTCGAGGTCTATGGCGCCGATGACGCGCAGC2160               CGAAGTTCGTGCAGGACTTCGTCGCTGCCTGGGACAAGGTGATGAACCTCGACAGGTTCG2220               ACGTGCGCTGATTCG2235                                                            (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2221 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        AGGAATGCTGTGCCCGAGCAACACCCACCCATTACAGAAACCACCACCGGAGCCGCTAGC60                 AACGGCTGTCCCGTCGTGGGTCATATGAAATACCCCGTCGAGGGCGGCGGAAACCAGGAC120                TGGTGGCCCAACCGGCTCAATCTGAAGGTACTGCACCAAAACCCGGCCGTCGCTGACCCG180                ATGGGTGCGGCGTTCGACTATGCCGCGGAGGTCGCGACCAGTCGACTTGACGCCCTGACG240                CGGGACATCGAGGAAGTGATGACCACCTCGCAGCCGTGGTGGCCCGCCGACTACGGCCAC300                TACGGGCCGCTGTTTATCCGGATGGCGTGGCACGCTGCCGGCACCTACCGCATCCACGAC360                GGCCGCGGCGGCGCCGGGGGCGGCATGCAGCGGTTCGCGCCGCTTAACAGCTGGCCCGAC420                AACGCCAGCTTGGACAAGGCGCGCCGGCTGCTGTGGCCGGTCAAGAAGAAGTACGGCAAG480                AAGCTCTCATGGGCGGACCTGATTGTTTTCGCCGGCAACCGCTGCGCTCGGAATCGATGG540                GCTTCAAGACGTTCGGGTTCGGCTTCGGGCGTCGACCAGTGGGAGACCGATGAGGTCTAT600                TGGGGCAAGGAAGCCACCTGGCTCGGCGATGACGGTTACAGCGTAAGCGATCTGGAGAAC660                CCGCTGGCCGCGGTGCAGATGGGGCTGATCTACGTGAACCCGGAGGCGCCGAACGGCAAC720                CCGGACCCCATGGCCGCGGCGGTCGACATTCGCGAGACGTTTCGGCGCATGGCCATGAAC780                GACGTCGAAACAGCGGCGCTGATCGTCGGCGGTCACACTTTCGGTAAGACCCATGGCGCC840                GGCCCGGCCGATCTGGTCGGCCCCGAACCCGAGGCTGCTCCGCTGGAGCAGATGGGCTTG900                GGCTGGAAGAGCTCGTATGGCACCGGAACCGGTAAGGACGCGATCACCAGCGGCATCGAG960                GTCGTATGGACGAACACCCCGACGAAATGGGACAACAGTTTCCTCGAGATCCTGTACGGC1020               TACGAGTGGGAGCTGACGAAGAGCCCTGCTGGCGCTTGGCAATACACCGCCAAGGACGGC1080               GCCGGTGCCGGCACCATCCCGGACCCGTTCGGCGGGCCAGGGCGCTCCCCGACGATGCTG1140               GCCACTGACCTCTCGCTGCGGGTGGATCCGATCTATGAGCGGATCACGCGTCGCTGGCTG1200               GAACACCCCGAGGAATTGGCCGACGAGTTCCGCAAGGCCTGGTACAAGCTGATCCACCGA1260               GACATGGGTCCCGTTGCGAGATACCTTGGGCCGCTGGTCCCCAAGCAGACCCTGCTGTGG1320               CAGGATCCGGTCCCTGCGGTCAGCACGACCTCGTCGGCGAAGCAGATTGCCAGCCTTAAG1380               AGCCAGATCCGGGCATCGGGATTGACTGTCTCACAGCTAGTTTCGACCGCATGGGCGGCG1440               GCGTCGTCGTTCCGTGGTAGCGACAAGCGCGGCGGCGCCAACGGTGGTCGCATCCGCCTG1500               CAGCCACAAGTCGGGTGGGAGGTCAACGACCCCGACGGATCTGCGCAAGGTCATTCGCAC1560               CCTGAAGAGATCCAGGAGTCATTCACTCGGCGCGGGAACATCAAAGTGTCCTTCGCCGAC1620               CTCGTCGTGCTCGGTGGCTGTGCGCCACTAGAGAAAGCAGCAAAGGCGGCTGGCCACAAC1680               ATCACGGTGCCCTTCACCCCGGGCCCGCACGATGCGTCGCAGGAACAAACCGACGTGGAA1740               TCCTTTGCCGTGCTGGAGCCCAAGGCAGATGGCTTCCGAAACTACCTCGGAAAGGGCAAC1800               CGTTGCCGGCCGAGTACATCGCTGCTCGACAAGGCGAACCTGCTTACGCTCAGTGCCCCT1860               GAGATGACGGTGCTGGTAGGTGGCCTGCGCGTCCTCGGCGCAAACTACAAGCGCTTACCG1920               CTGGGCGTGTTCACCGAGGCCTCCGAGTCACTGACCAACGACTTCTTCGTGAACCTGCTC1980               GACATGGGTATCACCTGGGAGCCCTCGCCAGCAGATGACGGGACCTACCAGGGCAAGGAT2040               GGCAGTGGCAAGGTGAAGTGGACCGGCAGCCGCGTGGACCTGGTCTTCGGGTCCAACTCG2100               GAGTTGCGGGCGCTTGTCGAGGTCTATGCGCCGATGACGCGGCAGGCGAAGTTCGTGACA2160               GGATTCGTCGCTGCGTGGGACAAGGTGATGAACCTCGACAGGTTCGACGTGCGCTGATTC2220               G2221                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        TCGGACCATAACGGCTTCCTGTTGGACGAG30                                               (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        AATCTGCTTCGCCGACGAGGTCGTGCTGAC30                                               (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        CACCCCGACGAAATGGGACAACAGTTTCCT30                                               (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        GGGTCTGACAAATCGCGCCGGGCAAACACC30                                               (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 740 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        ValProGluGlyHisProProIleThrGluThrThrThrGlyAlaAla                               151015                                                                         SerAsnGlyCysProValValGlyHisMetLysTyrProValGluGly                               202530                                                                         GlyGlyAsnGlnAspTrpTrpProAsnArgLeuAsnLeuLysValLeu                               354045                                                                         HisGlnAsnProAlaValAlaAspProMetGlyAlaAlaPheAspTyr                               505560                                                                         AlaAlaGluValAlaThrIleAspValAspAlaLeuThrArgAspIle                               65707580                                                                       GluGluValMetThrThrSerGlnProTrpTrpProAlaAspTyrGly                               859095                                                                         HisTyrGlyProLeuPheIleArgMetAlaTrpHisAlaAlaGlyThr                               100105110                                                                      TyrArgIleHisAspGlyArgGlyGlyAlaGlyGlyGlyMetGlnArg                               115120125                                                                      PheAlaProLeuAsnSerTrpProAspAsnAlaSerLeuAspLysAla                               130135140                                                                      ArgArgLeuLeuTrpProValLysLysLysTyrGlyLysLysLeuSer                               145150155160                                                                   TrpAlaAspLeuIleValPheAlaGlyAsnCysAlaLeuGluSerMet                               165170175                                                                      GlyPheLysThrPheGlyPheGlyPheGlyArgValAspGlnTrpGlu                               180185190                                                                      ProAspGluValTyrTrpGlyLysGluAlaThrTrpLeuGlyAspGlu                               195200205                                                                      ArgTyrSerGlyLysArgAspLeuGluAsnProLeuAlaAlaValGln                               210215220                                                                      MetGlyLeuIleTyrValAsnProGluGlyProAsnGlyAsnProAsp                               225230235240                                                                   ProMetAlaAlaAlaValAspIleArgGluThrPheArgArgMetAla                               245250255                                                                      MetAsnAspValGluThrAlaAlaLeuIleValGlyGlyHisThrPhe                               260265270                                                                      GlyLysThrHisGlyAlaGlyProAlaAspLeuValGlyProGluPro                               275280285                                                                      GluAlaAlaProLeuGluGlnMetGlyLeuGlyTrpLysSerSerTyr                               290295300                                                                      GlyThrGlyThrGlyLysAspAlaIleThrSerGlyIleGluValVal                               305310315320                                                                   TrpThrAsnThrProThrLysTrpAspAsnSerPheLeuGluIleLeu                               325330335                                                                      TyrGlyTyrGluTrpGluLeuThrLysSerProAlaGlyAlaTrpGln                               340345350                                                                      TyrThrAlaLysAspGlyAlaGlyAlaGlyThrIleProAspProPhe                               355360365                                                                      GlyGlyProGlyArgSerProThrMetLeuAlaThrAspLeuSerLeu                               370375380                                                                      ArgValAspProIleTyrGluArgIleThrArgArgTrpLeuGluHis                               385390395400                                                                   ProGluGluLeuAlaAspGluPheAlaLysAlaTrpTyrLysLeuIle                               405410415                                                                      HisArgAspMetGlyProValAlaArgTyrLeuGlyProLeuValPro                               420425430                                                                      LysGlnThrLeuLeuTrpGlnAspProValProAlaValSerHisAsp                               435440445                                                                      LeuValGlyGluAlaGluIleAlaSerLeuLysSerGlnIleArgAla                               450455460                                                                      SerGlyLeuThrValSerGlnLeuValSerThrAlaTrpAlaAlaAla                               465470475480                                                                   SerSerPheArgGlySerAspLysArgGlyGlyAlaAsnGlyGlyArg                               485490495                                                                      IleArgLeuGlnProGlnValGlyTrpGluValAsnAspProAspGly                               500505510                                                                      AspLeuArgLysValIleArgThrLeuGluGluIleGlnGluSerPhe                               515520525                                                                      AsnSerAlaAlaProGlyAsnIleLysValSerPheAlaAspLeuVal                               530535540                                                                      ValLeuGlyGlyCysAlaAlaIleGluLysAlaAlaLysAlaAlaGly                               545550555560                                                                   HisAsnIleThrValProPheThrProGlyArgThrAspAlaSerGln                               565570575                                                                      GluGlnThrAspValGluSerPheAlaValLeuGluProLysAlaAsp                               580585590                                                                      GlyPheArgAsnTyrLeuGlyLysGlyAsnProLeuProAlaGluTyr                               595600605                                                                      MetLeuLeuAspLysAlaAsnLeuLeuThrLeuSerAlaProGluMet                               610615620                                                                      ThrValLeuValGlyGlyLeuArgValLeuGlyAlaAsnTyrLysArg                               625630635640                                                                   LeuProLeuGlyValPheThrGluAlaSerGluSerLeuThrAsnAsp                               645650655                                                                      PhePheValAsnLeuLeuAspMetGlyIleThrTrpGluProSerPro                               660665670                                                                      AlaAspAspGlyThrTyrGlnGlyLysAspGlySerGlyLysValLys                               675680685                                                                      TrpThrGlySerArgValAspLeuValPheGlySerAsnSerGluLeu                               690695700                                                                      ArgAlaLeuValGluValTyrGlyAlaAspAspAlaGlnProLysPhe                               705710715720                                                                   ValGlnAspPheValAlaAlaTrpAspLysValMetAsnLeuAspArg                               725730735                                                                      PheAspValArg                                                                   740                                                                            __________________________________________________________________________ 

What is claimed is:
 1. A method for determining the susceptibility of a strain of M. tuberculosis to isoniazid comprising employing the technique of restriction length polymorphism analysis to determine whether a NciI-MspI restriction site is absent in the DNA of said strain at the codon corresponding to codon 463 of the M. tuberculosis katG gene consensus sequence depicted in FIG. 1 (SEQ ID NO:1), wherein said absence is indicative of an INH-resistant strain.
 2. The method of claim 1 which comprises the steps of:(a) amplifying a portion of the katG gene of an M. tuberculosis isolate to yield a detectable amount of DNA comprising a plurality of NciI-MspI restriction sites; (b) cleaving the amplified DNA with a restriction endonuclease at said sites to yield DNA fragments; and (c) employing the techniques of gel electrophoresis to determine whether the number and location of the DNA fragments is indicative of the absence of an NciI-MspI restriction site at codon 463 of said katG gene, wherein said absence is indicative of an INH resistant strain of M. tuberculosis in said isolate.
 3. The method of claim 2 wherein the amplified DNA comprises 4 NciI-MspI restriction sites prior to cleavage.
 4. The method of claim 1 wherein said DNA is amplified employing two oligonucleotide primers of the sequences (SEQ ID NO:5) and (SEQ ID NO:6) or subunits thereof, in a polymerase chain reaction to yield a 1435 base pair subunits of the katG gene.
 5. The method of claim 1 wherein the technique of polymerase chain reaction (PCR) is employed to amplify DNA from the katG gene of the isolate of M. tuberculosis to be assayed.
 6. An oligonuclcotidc selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, and subunits thereof which subunits are effective for the amplification of a region incorporating codon 463 of M. tuberculosis katG gene.
 7. The oligonucleotide of claim 6 selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. 